PROJECT SUMMARY Neurodevelopmental psychiatric disorders including intellectual disability (ID), autism (ASD) and schizophrenia (SZ) are often comorbid with neurological disorders, such as epilepsy. However, the biological substrates of such comorbidity are poorly understood. Understanding these molecular substrates could provide insight into the pathogenesis of these disorders as well as into fundamental neurobiological processes. One promising strategy is to investigate inherited, monogenic neurodevelopmental syndromes comorbid with epilepsy. Mutations in a number of genes have recently been discovered which cause neurodevelopmental disorders comorbid with epilepsy. Interestingly, the majority of such genes have putative functions at synapses and in dendrites, prompting the hypothesis that synaptic connectivity dysfunctions could be key for both types of disorders. Notably, mutations in a disproportionately large number of synaptic adhesion molecules are associated with neuropsychiatric disorders, underscoring the importance of understanding their neuronal functions. A significant fraction of these genes encode members of the neurexin and contactin family. Here we propose to investigate the synaptic and dendritic functions of a prominent representative of this family, CNTNAP2, mutations in which cause monogenic syndromes of ID, ASD, and SZ comorbid with epilepsy. Because Cntnap2 modulates synapse structure and function, here we propose to investigate novel molecular mechanism underlying Cntnap2 functions newly discovered by us, such as glutamate receptor trafficking, interneuron dendrite arborization, and paracrine signaling by ectodomain shedding. Based on our preliminary studies we hypothesize that Cntnap2 plays crucial roles in AMPAR trafficking and in the maintenance of synapto-dendritic architecture through its protein interaction network. We will test this hypothesis by employing several novel and cutting-edge methodologies such as superresolution and multi-photon imaging, LC-MS/MS proteomics, and CRISPR/Cas9-engineered iPSC-derived neurons (iN), and by integrating mechanistic studies in neuronal cultures with human iNs and mouse models. We will pursue the following Specific Aims: 1) Regulation of AMPAR trafficking by Cntnap2 and its protein partners; 2) Control of spine architecture and interneuron dendrite arborization by Cntnap2 and its partners; 3) Mechanisms of paracrine signaling by Cntnap2 extracellular domain shedding. Data generated will provide novel insight into signaling by Cntnap2, the regulation of synaptic circuits in the brain shedding new light on glutamate receptor trafficking by adhesion molecules, formation of neurotransmitter receptor intracellular aggregates, nanoscopic localization of synaptodendritic molecules, interneuron-specific maintenance of dendritic arborization, control of cortical E/I balance, as well as paracrine signaling by ectodomain shedding. Proposed studies will also uncover mechanisms potentially relevant for the pathogenesis of neurodevelopmental disorders.